Background Natural killer(NK) cells, as key effector cells of innate immunity, can recognize and kill tumor cells without prior sensitization, holding significant potential in cellular immunotherapy. Companion dogs develop spontaneous tumors exhibiting pathological types, molecular characteristics, and immune systems highly analogous to humans, indicating substantial promise for utilizing cellular immunotherapy as a novel therapeutic approach for canine malignancies. However, the intricate tumor microenvironment constrains NK cell activity and impedes their function, while technical bottlenecks such as low viability and limited expansion efficiency also fail to meet clinical demands. This study optimized an expansion system based on “multiple stimulations of peripheral blood mononuclear cells (PBMCs) with engineered K562 feeder cells,” evaluated the anti-tumor functionality of the derived canine NK cells, and, for the first time, integrated metabolomics technology to decipher the metabolic reprogramming mechanisms underlying NK cell-tumor cell interactions. Results PBMCs isolated from healthy donor dogs yielded 3.312 × 109 CD3−CD21−CD94+ canine NK cells after 14 days of culture, while those from tumor-bearing dogs yielded 9.197 × 108 cells. Cultured canine NK cells showed significant upregulation in the expression of perforin, granzyme B, and NKp46. Twice-stimulated NK cells exhibited enhanced tumor cell killing compared to single stimulation, with maximum killing efficiency at 40:1 (98.00% ± 0.42 (n = 3)) and 20:1 (72.00% ± 2.02 (n = 3)). Notably, 40:1 induced 5.01% ± 0.04 (n = 3) normal cell cytotoxicity. IFN-γ secretion increased significantly post-co-culture across all tumor lines, while perforin and granzyme B displayed time-dependent secretion dynamics. LC-MS/MS-based metabolomics identified 199 differentially abundant metabolites in the NK-D17 interaction, with 44 up-regulated and 155 down-regulated. Dysregulated pathways included glycerophospholipid metabolism, triacylglycerol metabolism, vitamin B6 metabolism, alanine, aspartate and glutamate metabolism, and tryptophan metabolism. Conclusions The optimized canine NK cell culture system developed in this study enables the efficient expansion of CD3−CD21−CD94+ canine NK cells and confirms their cytotoxicity against K562, D17, and CMT1211 tumor cell lines. The tryptophan metabolism pathway may represent a key mechanism for the functional inhibition of canine NK cells, meriting further investigation to elucidate the mechanisms underlying NK cell-tumor cell interactions. These experimental results provide a foundation for developing canine NK cell-based therapies.

Metabolomic profiling and anti-tumor function of canine NK cells in tumor interaction

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2026年2月28日

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Shi He,
Shufeng Wu,
Laiyu Chen,
Yugu Li,
Bingyu Wang

摘要

Background

Natural killer(NK) cells, as key effector cells of innate immunity, can recognize and kill tumor cells without prior sensitization, holding significant potential in cellular immunotherapy. Companion dogs develop spontaneous tumors exhibiting pathological types, molecular characteristics, and immune systems highly analogous to humans, indicating substantial promise for utilizing cellular immunotherapy as a novel therapeutic approach for canine malignancies. However, the intricate tumor microenvironment constrains NK cell activity and impedes their function, while technical bottlenecks such as low viability and limited expansion efficiency also fail to meet clinical demands. This study optimized an expansion system based on “multiple stimulations of peripheral blood mononuclear cells (PBMCs) with engineered K562 feeder cells,” evaluated the anti-tumor functionality of the derived canine NK cells, and, for the first time, integrated metabolomics technology to decipher the metabolic reprogramming mechanisms underlying NK cell-tumor cell interactions.

Results

PBMCs isolated from healthy donor dogs yielded 3.312 × 10⁹ CD3⁻CD21⁻CD94⁺ canine NK cells after 14 days of culture, while those from tumor-bearing dogs yielded 9.197 × 10⁸ cells. Cultured canine NK cells showed significant upregulation in the expression of perforin, granzyme B, and NKp46. Twice-stimulated NK cells exhibited enhanced tumor cell killing compared to single stimulation, with maximum killing efficiency at 40:1 (98.00% ± 0.42 (n = 3)) and 20:1 (72.00% ± 2.02 (n = 3)). Notably, 40:1 induced 5.01% ± 0.04 (n = 3) normal cell cytotoxicity. IFN-γ secretion increased significantly post-co-culture across all tumor lines, while perforin and granzyme B displayed time-dependent secretion dynamics. LC-MS/MS-based metabolomics identified 199 differentially abundant metabolites in the NK-D17 interaction, with 44 up-regulated and 155 down-regulated. Dysregulated pathways included glycerophospholipid metabolism, triacylglycerol metabolism, vitamin B6 metabolism, alanine, aspartate and glutamate metabolism, and tryptophan metabolism.

Conclusions

The optimized canine NK cell culture system developed in this study enables the efficient expansion of CD3⁻CD21⁻CD94⁺ canine NK cells and confirms their cytotoxicity against K562, D17, and CMT1211 tumor cell lines. The tryptophan metabolism pathway may represent a key mechanism for the functional inhibition of canine NK cells, meriting further investigation to elucidate the mechanisms underlying NK cell-tumor cell interactions. These experimental results provide a foundation for developing canine NK cell-based therapies.